Antioxidant, anti‐inflammatory, and anticancer function of Engleromyces goetzei Henn aqueous extract on human intestinal Caco‐2 cells treated with t‐BHP

Abstract High body mass index (high BMI, obesity) is a serious public health problem, and “obesity‐induced oxidative stress, inflammation, and cancer” have become modern epidemic diseases. We carried out this study to explore a functional beverage that may protect against obesity‐induced diseases. The Engleromyces goetzei Henn herbal tea is such a candidate. For this study, we carried out LC–MS analysis of E. goetzei Henn aqueous extract (EgH‐AE); then used the Caco‐2 cell line for the model cells and treated the cells with t‐BHP to form an oxidative stress system. An MTT assay was used for testing the biocompatibility and cytoprotective effects; reactive oxygen species and malondialdehyde determination was used for evaluating the antioxidative stress effect; TNF‐α and IL‐1β were used for observing the anti‐inflammatory effect, and 8‐OHdG for monitoring anticancer activity. The results of this study demonstrate that the EgH‐AE has very good biocompatibility with the Caco‐2 cell line and has good cytoprotective, antioxidant, anti‐inflammatory, and anticancer properties. It is clear that EgH‐AE, a kind of ancient herbal tea, may be used to develop a functional beverage that can be given to people with a high BMI to protect against obesity‐induced diseases.

about 5,019,360 people in 2019 (The Institute for Health Metrics and Evaluation, 2020). The prevalence of obesity is also very high.
In terms of pathogenesis, oxidative stress is usually an upstream event of inflammation and cancer (Reuter et al., 2010). Reactive oxygen species (ROS) can induce the synthesis and release of inflammatory factors such as TNFα and IL-1β, by activating the NFκB signaling pathway. In addition, high levels of ROS in cells attack guanine bases in DNA easily and form 8-hydroxydeoxyguanosine (8-OHdG), and 8-OHdG can induce the occurrence of various cancers.
Therefore, in the face of the above diseases, in addition to clinical treatment, appropriate use of antioxidant functional foods could also form a very important strategy for the prevention and treatment of obesity-induced diseases.
At this stage, antioxidant functional foods are mainly derived from plants, such as grapes, blueberries, kiwifruit, green tea, etc.; these foods are rich in antioxidants, such as blueberries rich in anthocyanins, kiwi fruit rich in vitamin C, green tea rich in tea polyphenols, and these substances play a significant role in maintaining the health of the body and slowing down the oxidation of the body (Ramarathnam et al., 1995). At the same time, edible and medicinal fungi are a kind of functional food that is rich in nutrients and has vigorous antioxidant activity; at present, edible and medicinal dualuse fungi have become another important source of antioxidant functional food (Mwangi et al., 2022).
Engleromyces goetzei Henn. (EgH) (Fungal, Ascomycetes, Hypocreaceae, Hypocrea), the shape is irregularly spherical, 2-20 cm in diameter, nearly smooth in appearance with fine black spots, the whole is pink or light flesh, anaphase becomes milky white, grayish to brown, the inside is light red to gray-white, solid, hard after drying, interwoven by short and irregular branched mycelium, containing a large number of spherical oil droplets, mature bamboo fungus surface is often uneven, or with irregular protrusions, the fungus meat has a slightly bitter taste after chewing (Yunnan Institute of Botany, 1975). In China, EgH parasitic on high mountain bamboo poles and matures in the rainy season from July to August, this fungus is mainly distributed between 25-30° N and 98-103° E, that is, Northwest Yunnan, Southwest Sichuan, and Southeast Tibet. The terrain of this region is steep, and many mountains are arranged in parallel in a north-south direction. It is one of the regions with the highest biodiversity in Asia and even in the world. EgH is also a native functional food and medicine with a long tradition of use in Yunnan, Sichuan, Tibet, and other places. It is often used by local residents to make tea with boiling water and is used to treat infection, inflammation, and cancer (Jikai et al., 2002;. An earlier experimental study found that EgH has a broad-spectrum antibacterial effect (Yunnan Institute of Botany, 1975). Zhang et al. (2019) found experimentally that EgH has an antiproliferation effect. Wang's study  found that We, therefore, considered exploring whether the traditional remedy using EgH tea can be used to prevent and treat obesityinduced oxidative stress, inflammation, and cancer. In this study, we investigated the antioxidant, anti-inflammatory, and anticancer effects of E. goetzei Henn aqueous extract (EgH-AE) on Caco-2 cells treated with t-BHP. The results support the possibility that EgH-AE may be developed as a functional beverage to protect the health of obese patients. Interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) kits were purchased from Wuhan Boster Bioengineering Co., Ltd. ROS kit was from Jiangsu Jingmei Biological Technology Co., Ltd.

| Preparation of Engleromyces goetzei Henn extracts
Natural E. goetzei Henn (EgH) were harvested from Yunling-Lasha Mountain Nature Reserve, Yunnan Province, China during the summer of 2021. Figure 1 shows a sample image of EgH and the preparation protocol of the EgH extracts.
The sun-dried EgH sample was smashed and passed through a 100-mesh sieve. Place 50 g of EgH powder into a 500 mL conical flask, and add 500 mL of petroleum ether according to a materialto-liquid ratio of 1:10. At 20°C, 60% power, 40 Hz ultrasonic extraction for 1 h. After standing in the dark for 24 h, the supernatant was decanted using a pipette, suction filtered, and the filtrate concentrated by rotary evaporation. The remains were freeze-dried to obtain the petroleum ether extract of EgH. The filter residue was dried at 40°C, and the above operation was repeated using ethyl acetate, n-butanol, 95% ethanol, and distilled water to successively extract filter residue, the ethyl acetate extract, n-butanol extract, 95% ethanol extract, and aqueous extract of EgH were obtained, respectively, and stored at 4°C for future use.

| DPPH assay
The DPPH radical scavenging assay was performed according to a previous report (Brand-Williams et al., 1995). The EgH aqueous extract (EgH-AE) was diluted with absolute ethanol to form different concentrations of solvent (15, 10, 5, 2.5, 1.25, and 0.625 mg/mL). 50 mg/L DPPH absolute ethanol solvent was blended in the ratio of 1:1 and left to stand at 37°C for 30 min in the dark. Absolute ethanol was used as the control. Absorbance of the reaction mixtures was recorded at 517 nm. Five replicates, one sample control, and one blank control experiment were set up for each concentration. In addition, a standard curve was drawn with a DPPH of 5-100 mg/L. The DPPH radical scavenging activity of the EgH extract was calculated following the equation:

| ABTS assay
The ABTS radical scavenging assay was performed according to a previous report (Miller et al., 1993). The EgH-AE was diluted with absolute ethanol to form different concentrations of solvent (15, 10, DPPH radical scavenging activity = Abs of control − Abs of sample Abs of control × 100 % . F I G U R E 1 Photographs of EgH and the sample extraction procedure. a1 and a2 (our image), (b) Preparation protocol for EgH extraction, the EgH images are ours. 5, 2.5, 1.25, and 0.625 mg/mL). ABTS radical ethanol solvent was blended with each sample in the ratio of 1:1 and left to stand at room temperature for 6 min. Absolute ethanol was used as the control. Absorbance of the reaction mixtures was recorded at 734 nm.
Six replicates, one sample control, and one blank control experiment were set up for each concentration. The ABTS radical scavenging activity of the EgH extract was calculated as follows:

| Measurement of total phenolic, flavonoid, and polysaccharide content
The total phenolic content was quantified using Folin-Ciocalteu reagent and gallic acid standard (Ainsworth & Gillespie, 2007). Briefly, 1 g/mL of EgH-AE was mixed in a test tube containing 5 mL 10% Folin-Ciocalteu reagent. The mixture was then allowed to react for 3-8 min, after which 4 mL of 7.5% Na 2 CO 3 was added. The mixture was then placed in a dark room for 1 h, and the absorbance was measured at 765 nm. The total phenolic content is expressed as gallic acid equivalents (μg GAE/mg).
To analyze total flavonoid content in EgH-AE (Zhishen et al., 1999), 1 g/mL of each sample was mixed with 300 μL of 5% (w/v) NaNO 2 and 300 μL of 10% Al(NO 3 ) 3 , The mixture was then allowed to react for 6 min, after which 1 mL of 1 mol/L NaOH and 3.4 mL of 30% ethyl alcohol was added. The mixture was then placed in a dark room for 15 min, and the absorbance was measured at 510 nm. The total flavonoid content is expressed as rutin equivalents (μg RE/mg).
To analyze the total polysaccharide content of EgH-AE (Dubois et al., 1951), 1 g/mL of each sample was mixed with 1 mL of 5% phenol and 5 mL of 1.84 g/mL H 2 SO 4 . The mixture was then placed in a dark room for 30 min, and the absorbance was measured at 490 nm.
The total polysaccharide content is expressed as glucose equivalents (μg GE/mg).

LC-MS was performed in electrospray ionization (ESI) mode with
a liquid chromatography instrument (U3000; Thermo Fisher) combined with a mass spectrometer (QE Plus; Thermo Fisher). Accurately add 300 μL of water prepared with 2-chlorol-phenylalanine (4 ppm) solution for redissolution EgH-AE. The EgH-AE was centrifuged at 17,435 g at 4°C for 10 min and filtered through a 0.22 μm membrane.

| Cell culture
Caco-2 cells (human colon adenocarcinoma cells) were purchased from Kunming Cell Bank of Type Culture Collection, Chinese Academy of Sciences. The cells were inoculated into a 75 cm 2 cell culture flask containing 15 mL culture medium, placed in a 37°C, 5% CO 2 , and high humidity incubator, and the culture medium was changed every 24 h. The culture medium comprised 84% DMEM high glucose medium, 15% fetal bovine serum, 1% penicillin (1000 U/ mL), and 1% streptomycin (1000 μ g/mL). The cells were subcultured after 80%-90% of the bottom of the bottle.

| MTT assay
Also called a cell viability assay, this was carried out by the 3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Mosmann, 1983). Briefly, the Caco-2 cells were seeded into a 96well cell culture plate with 1 × 10 5 cells/well, and treated with dif- The concentration of Caco-2 cells was adjusted to 5 × 10 5 cells/ mL, inoculated 2.0 mL cell suspension in each well of the 6-well plate, and placed in the incubator for culture, changing the solvent every 12 h. After 2 days, discard the cell culture medium, add 1 mL PBS to each well to clean it, and repeat twice. The Caco-2 cells were then preprotected for 12 h with different concentrations (0.0, 1.0, 5.0, and 10 mg/mL) EgH-AE and 0.5 mg/mL Vit C. After that, the cells were treated with 2 μmoL/mL t-BHP. The EgH-AE, t-BHP, and Vit C were diluted with DMEM medium. Each group was equipped with six experimental holes. After 5 h, the cell viability (%) was determined using a commercial kit (Cat. No. 16H12B56; Shanghai Enzyme Link Biotechnology Co., Ltd.). All samples were prepared according to the instructions for the use of the MTT kit.

| Statistical analyses
All data in this study were analyzed by one-way ANOVA, followed by an LSD test, the LSD test at 5% precision level was used to ascertain the significance and nonsignificance of different treatment and control groups. Data were processed with GraphPad Prism 7 software and results were expressed as the mean and standard error of the mean (mean ± SD).

| Free radical scavenging activity of EgH extracts
In this study, we used DPPH and ABTS radical scavenging assays to evaluate the antioxidant activity of the EgH extracts with different solvents. The results are presented in Figure 2 and Table 1.
As shown in Figure 2,

| In vitro biocompatibility of EgH-AE
Biocompatibility, which is sometimes called "cytotoxicity", is the most commonly used term to describe the appropriate biosafety requirements of agents or biomaterials. Their presence in bodies will not cause cell damage or affect the function of cells. Biocompatibility is also described as the ability of agents or biomaterials to have an appropriate host response in a specific application. One of the most common methods for testing biocompatibility is the MTT assay (Aoki & Saito, 2020;Mastrogiovanni et al., 2019;Stan et al., 2019).
To assess biocompatibility, the Caco-2 cells were treated with different concentrations of EgH-AE for 24 h. After 24 h of exposure, MTT test results ( Figure 5) showed that cell viability remained statistically unchanged when compared to the untreated cells (0.00 mg/

mL group). The viability of the cells incubated in the presence of the
EgH-AE showed a slight change of 0.0%-10% for all of the tested doses, proving that the extract had no harmful effects on the intestinal cells.

| Cytoprotective effect of EgH-AE against t-BHP-induced damage
We evaluated the cytoprotective effect of EgH-AE against oxidative stress induced with t-BHP, by MTT assay at a concentration of 2 μmol/mL for 5 h. The results of cell viability for the different groups are shown in Figure 6.
It is clear that: (1)  means that t-BHP treatment can badly damage Caco-2 cells; (2) Compared with the t-BHP only group, the cell viability in the Vit C + t-BHP group increased significantly (p < .001), indicating that the positive reference agent, Vit C, has a good cytoprotective effect; (3) Compared with the t-BHP only group, the cell viability in the EgH-AE + t-BHP groups also increased significantly (p < .01, p < .001, p < .001), indicating that the EgH-AE groups achieved a positive cytoprotective effect, and this effect is very closely dose-related, a result that is similar to other studies found in the literature (Antognoni et al., 2020;Bedoya-Ramírez et al., 2017;Dai et al., 2020).
The effect of different treatments on the morphology of Caco-2 cells is shown in Figure 7. The cells of the CK group grow well, the adherent wall is firm, the cells are tightly connected in a polygonal shape, the size is uniform, the edges are distinct, and the paving stone-like single-layer mosaic is arranged. After 5 h of 2 μmoL/ mL t-BHP treatment, Caco-2 cells increased the intercellular space, the morphology of the cells became blurred, and the cells began to fall off in large quantities. After the cells were preprotected by different concentrations of EgH-AE, the cell morphology was less and less affected by t-BHP with the increase of EgH-AE treatment concentration, and the cell morphology of the 10 g/L EgH-AE + t-BHP group was similar to that of the CK group. At the same time, after the cells were preprotected by Vit C, the effects caused by t-BHP were primarily avoided, but the protection degree of 10 g/L EgH-AE could not be achieved. Figure 8 illustrates the main biomarkers for indicating cellular oxidative stress and inflammation. In this study we chose: ROS and MDA as the biomarkers for proving antioxidant activity (Bartolomei et al., 2021;Lin et al., 2020;Wang et al., 2020); TNFα and IL-1β as the biomarkers for proving anti-inflammation activity (Lameirão et al., 2020;Mastrogiovanni et al., 2019;Park et al., 2022); and 8-OHdG as the biomarker for proving anticancer activity (Jee et al., 2020;Reuter et al., 2010;Tiwari & Mishra, 2017;Yadav et al., 2016), although it can be also be used as a biomarker of oxidative stress.

| Antioxidant activity of EgH-AE in Caco-2 cells
The results of ROS determination are shown in Figure 9a: (1) The levels of ROS in Caco-2 cells treated with 2 μmoL/mL t-BHP, after 5 h, F I G U R E 3 Overview of marine-derived polysaccharides in alleviating oxidative stress-mediated diseases (cited from Zhong et al., 2019).

| Anti-inflammatory activity of EgH-AE
The results for TNFα levels are shown in Figure 10a: (1)   EgH-AE is a very good anti-inflammatory agent, and this effect also has a dose-effect relationship.

| Anticancer activities of EgH-AE
8-OHdG is a special biomarker, because it is one of the endpoints of oxidative stress, but is also an initiator of carcinogenesis (Reuter et al., 2010). The ROS-derived DNA damage includes the generation of 8-hydroxyguanosine, the hydrolysis product of which is 8-hydroxydeoxyguanosine (8-OHdG). 8-OHdG is the most widely used indicator of a radical attack on DNA (Marnett, 2000;Wiseman & Halliwell, 1996). 8-OHdG is strongly implicated in carcinogenesis progression. For example, in breast carcinomas, levels of 8-OHdG have been reported to be 8-17-fold higher in primary breast tumors compared with healthy breast tissue (Musarrat et al., 1996;Valavanidis et al., 2009).
The results of 8-OHdG determination are shown in Figure 11: (1) The levels of 8-OHdG, in Caco-2 cells after 5 h of treatment with 2 μmoL/mL t-BHP increased significantly, in the t-BHP group compared with the blank control group (p < .001). This means that t-BHP can cause extensive DNA oxidative damage, which may then induce DNA base mismatch and sequence point mutation in Caco-2 cells; (2) Compared with the t-BHP group, levels of 8-OHdG in the Vit C group decreased significantly (p < .001), indicating that Vit C is protective against cancer; Compared with the 1.0 EgH-AE group, Vit C is a better anticancer agent (p < .05), but compared with the 10.0 EgH-AE group, Vit C is not as effective an anticancer agent (p < .01); (3) Compared with the t-BHP group, the levels of cellular 8-OHdG in each EgH-AE group decreased in a dose-dependent manner (ns, p < .001, p < .001), indicating that a higher dose of EgH-AE has very good anticancer properties, and this anticancer effect also has a dose-effect relationship.

| CON CLUS ION
In this study, we found that E. goetzei Henn aqueous extract (EgH-AE) is very biocompatible with the Caco-2 cell line, and has good cytoprotective, antioxidant, anti-inflammatory, and anticancer properties. The results of this study support the idea that EgH-AE, the historical herbal tea used in Yunnan province of China, may be used to develop a functional beverage that can be given to people with a high BMI to protect against obesity-induced diseases.

ACK N OWLED G M ENTS
This research was supported by the "Collaborative Innovation Center for Biodiversity and Conservation in the Three Parallel Rivers Region F I G U R E 1 0 Anti-inflammatory effect of EgH-AE in Caco-2 Cells. (a) The levels of cellular TNFα; (b) the levels of cellular IL-1β. The data were analyzed by one-way ANOVA and followed by an LSD test. Asterisks indicate comparison with the t-BHP group, *p < .05, **p < .01, ***p < .001; Well numbers mean comparison with the Vit C group, # p < .05, ## p < .01; well numbers indicate comparison with the Vit C group, # p < .05, ## p < .01, ### p < .001.

F I G U R E 11
Anticancer activity of EgH-AE in Caco-2 cells. The results of cellular 8-OHdG were analyzed by one-way ANOVA and followed by an LSD test. Asterisks indicate comparison with the t-BHP group, *p < .05, **p < .01, ***p < .001; well numbers indicate comparison with the Vit C group, # p < .05, ## p < .01, ### p < .001.
of China, Dali University, Dali, Yunnan, China" and "The Provincial Innovation Team of Biodiversity Conservation and Utility of the Three Parallel Rivers Region, Dali University, Dali, Yunnan, China".

FU N D I N G I N FO R M ATI O N
This research was funded by "Development of diagnostic instrument for oxidative inflammation: determination of blood ROS, MDA and urine 8-OHdG", grant number "202101BA070001-115".

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data used for this study are available on request through the corresponding author, although all the relevant data have been provided here.

E TH I C S S TATEM ENT
This research did not include biological agents of risk, or clinical trials with humans or animal experiments.